The general goal of these studies is to elucidate the mechanism by which thrombin potentiates the action of mitogens like epidermal growth factor (EGF) on cultured human vascular endothelial cells. From a physiological point of view it seems probable that this pattern of growth factor action is involved in endothelial regeneration following vascular injury. Our plan is to identify what thrombin does to the endothelial cell to make it more responsive to EGF, taking advantage of previous studies on thrombin-related and EGF-related cellular components in fibroblast-like cells where thrombin or EGF is mitogenic by itself. We will determine whether thombin must be internalized by endothelial cells and whether its proteolytic activity is necessary for potentiation of EGF action. If proteolysis is required, we will look for cleavages of individual cell surface or intracellular proteins using two-dimensional gels. The relationship of identified cleavages to potentiation by thrombin will be probed by analyzing the thrombin concentration and treatment time required for each event and by examining the ability of other proteases of limied action to cause potentiation and the cleavage in question. We will label endothelial thrombin "receptors" with photoreactive cross-linking derivatives of 125I-thrombin so we can locate them on gels and determine, for example, if they are cleaved by thrombin. Experiments will be conducted to determine if endothelial cells produce and release protease-nexin, a fibroblast component we recently identified that mediates much of the cellular binding of thrombin. To guide experiments on thrombin-mediated changes in EGF-related cellular components, we will determine whether the mitogenic action of EGF on endothelial cells requires internalization. Then we will examine the effects of thrombin treatment on binding, internalization and degradation of 125I-EGF, and internalization and "processing" of EGF receptors. Experiments will also be conducted to explore the effects of thrombin on steady-state phosphorylation of EGF receptors and certain other cellular components.